JP2698337B2 - Anti-villin monoclonal antibody that can be used for in vitro detection of malignant cells of gastrointestinal origin - Google Patents

Abstract

The invention relates to biologically pure human villin, characterised in that it essentially gives only a single band, with a molecular weight of 95 kD, while subjected to polyacrylamide gel electrophoresis, and characterised in that each COOH terminus comprises the following amino acid sequence: <IMAGE> The invention also relates to a peptide fragment of human villin, characterised in that it is recognised by antibodies which recognise biologically pure human villin, and to anti-villin antibodies.

Description

DETAILED DESCRIPTION OF THE INVENTION [0001] The present invention is directed to tumor cells in the human gastrointestinal tract.
The presence or absence of proliferation of malignant cells, particularly the presence or absence of proliferation of adenocarcinoma-type cells
material elements that can be used in the method for n vitro detection,
Especially human villiAntibodies to proteins, preferably monoclonal
Related to lonal antibody. [0002] The invention further relates to various uses, for example, human vagina.
Human Villin Protein from Cell Extract Containing Hair Protein
These when used to purifyMonocrona
AntibodyTo the effectReach. To identify several higher eukaryotic cell types
And the developmental stages of these cells, especially the differentiation stages
For trace studies, use of structural proteins as specific labels
The use of detection has already been proposed. Known label
For example, fibrous proteins such as vimentin,
There are ratines or nerve fiber proteins, some of which are
Cell type identification, from embryogenesis to final cell differentiation
Of cell development in soil and cell behavior on environmental media
(E.LAZARIDES, Nature,283, 249
(1980) and R. MOLL, Cell,31, 11 (1982)). [0004] Keratin is often found in epithelial and non-epithelial cells.
It is considered to be a particularly effective label that can identify However
However, this type of identification requires the source of the epithelial cells in question.
It turned out that it could not be definitely determined. [0005] However, the origin of some cell types has been confirmed.
In many cases, the need to detect
When examining biological samples that show vesicle growth, this increase
To identify or eliminate primary tumor cells present at the origin of the
To detect the presence of metastasis originating from cancer in the activated region or
Confirm the effects of chemotherapy and other therapies on metastatic cancer
Is gaining particular importance. Of course, anti-cancer treatment
Efficacy depends on accurate identification of tumor cells as primary cause of disease
The depth is deep. [0006] These observations suggest that
Of the digestive tract or kidney
Growth of tumor cells or cells derived from these cells
Detection of proliferation, preferably early detection, especially for these cells
Of particular importance for label identification. Accordingly, the present invention is particularly directed to mucous membranes of the digestive tract,
Normally present in the intestinal mucosa and the main differentiated type is absorbed cells or intestinal cells.
Maintains several major phenotypes of epithelial cells composed of vesicles
Is important for the detection of isolated tumor cells. [0008] The present invention also relates to a method for carrying these tumor cells.
Specific labels that can be detected whether or not
You. It is a particular object of the present invention that these labels may be used in tumors.
In the case of necrosis of a cell, especially the cell, detached from the cell, for example,
Detect label even when released into vasculature
You. Labels used within the scope of the present invention are
It is composed of all or part of the hair protein. Villous tongue
Protein is normally found in villous tissue of the digestive mucosa, especially the intestinal mucosa.
A protein of the order of 95,000 daltons
is there. Villin protein is activated in the presence of calcium ions.
Can bind to Kuching. [0011] Amino acid sequence differs slightly for each type of villi
However, the functional characteristics are maintained. Well-known spirits to date
Manufacturing technology substantially eliminates chicken and porcine villous proteins
To a pure state. However, these technologies
Cannot purify human villin protein. One of the best known villous proteins is
Chicken villous protein. This villous protein
Is composed of 854 amino acid sequences. Trypsi
Degradation of Protein Using Protein and Protease V-8
To cut 44Kd, 51Kd and 10Kd fragments.
Obtainable. This in the villous protein map
The relative positions of the fragments were determined by Paul MADSUDAIRA, MRC, Laboratory
of Molecular Biology, Cambridge, U.K. and John GLE
According to NNEY et al., J.B.C., 1981, 256, 8156-8161
Has been confirmed as. [0013] [Table 1] The COOH terminal of chicken villous protein
Amino acid sequence (779-854) or "head" ("peptide H
P ") is represented by the following equation. [0015] Embedded imageIn general, villous proteins are particularly
Border of the intestinal lumen wall observed on the surface of enterocytes reaching the stomach
Exists inside. Microvilli are end-stage differentiation organs of enterocytes
Nera. Villous proteins are particularly the microscopic villi
Localized during convergence. Villin protein is located in the cytoskeleton
Contribute to the configuration. Examining frozen sections by immunofluorescence
Forms a column that exposes the villous protein to the intestinal lining
Located at the apex of elongated cells and in the proximal tubule cells of the kidney
It was confirmed that it also existed in the vicinity of. In both cases
The region where the hair protein is located coincides with the brush border of the cell
You. However, various other villi of various other epithelia
Villus tans when they do not have highly organized brush borders
No protein was detected (Exp.Cel, A.BRETSCHER etc.)
l.res.135, 213 (1981) or A.LISS's book Membrane in
 Growth and Development, New
 York (1982), pp.89-105, H.REGGIO
Sentence). Furthermore, the villin protein is more likely to form
It was also found to be present in enterocytes in the early stage of development.
Was. The present invention has been obtained based on two findings.
It is. The villous protein is characteristic during tissue tumor formation in the gastrointestinal tract.
Colon cancer, one of the most frequent forms of cancer, and some
Is virtually systematic in kidney cancer types
But maintained in other organs (liver, ovary, lungs)
Primary tumor cells arising from early carcinogenesis of tissue localized to
It is never detected in. In addition, villous protein is a tumor in the gastrointestinal tract.
Early and later oncogenesis at all sites of cell development
In vivo, regardless of the differentiation status of the cells
It is possible. Formed in the digestive tract from the beginning in biological samples
The tumor cells and / or derivatives of the tumor cells in vit
ro Detection method of the present invention mainly for detecting
The characteristic of this biological sample is that
Have specific affinity or
Contact with a reagent that recognizes the gene to be loaded. The detection method of the present invention is based on
Tumor cells or primary cells of digestive organ or kidney origin
Tumor cells formed later in another part of the organism by the effect
All biological samples containing cells or necrosis of the cells
In some cases, the degradation products of various tumor cells, including
It can be applied to all biological samples to be encapsulated. This creature sun
The pull may be in any form, for example, obtained by biopsy.
Tissue or solid tumor fragments or malignant cells or
Is a liquid biological sample carrying fragments from said cells, such as whole blood
Or serum. According to an embodiment of the present invention,
The reagents used in the detection methods
Contains the region encoding the amino acid sequence of the COOH terminal.
It is composed of DNA having the sequence According to another embodiment of the present invention, a detection method is provided.
The technique used in immunization is an immunological technique,
Antibodies that can recognize villin protein
Consists of These antibodies can be prepared by any known method.
The body is labeled or villous protein or villi
Label when fixed to hair protein-containing cells
Immunoglobulins or other labeled proteins (eg,
Staphylococcus aureusCan be recognized by protein A)
You. A particularly suitable technique is that used in the tests described below.
This is a technique showing the results as described above. An object of the present invention is also to provide intestinal cells or the like cells.
Novel method for detecting villous proteins in microvilli
To provide a unique means (of course,
The use is not necessarily limited to the detection method in the present invention.
No). According to one feature of the invention, these means
Is the DNA corresponding to the complete mRNA of human villin protein or
Region encoding the amino acid sequence of human villin protein
A DNA fragment having a nucleotide sequence containing
Specific to the nucleotide sequence encoding the hair protein
May hybridize. The invention is particularly directed to the completeness of the human villin protein.
DNA corresponding to mRNA or COOH terminus of human villous protein
At least the nucleotide sequence encoding the amino acid
Using the cDNA fragment containing the part in the method.
Aim. This nucleotide sequence and the encoded
The corresponding sequences of the amino acids are shown below. [0029] Embedded image The nucleotide sequence used is human villi
For detecting mRNA or DNA encoding a protein
Used as a probe. Probes suitable for this type of detection are radioactive
Labeled by element or test mRNA or DNA
That are recognizable in a hybridized state with a preparation containing
Advantageously, it is labeled by a substance. According to the prior art, these probes and the
The biological sample containing cells is separated from the nucleotides of the probe.
The sequence and the complementary sequence arbitrarily contained in the test sample are
Contact or nuclei under conditions that allow
Contact directly with acid. A probe containing a 200 base pair insert was
Was used to test the reproducibility of detection. Novel products within the scope of the present invention
Another feature of these nucleotide probes that make up
Differentiation status of cells and / or cells expressing villous protein
Villi at different stages of maturation of cells of renal or intestinal origin
Used to study mRNA expression of proteins
And Therefore, the obtained probe is a human gene
Number and tissue of villous protein genes isolated from
And the structure can be determined. According to the present invention, the human villi protein
Isolation of the nucleotide sequence encoding the end
Done: -A cell line expressing villin protein using known techniques
Prepare complete mRNA (messenger RNA) Construct a cDNA (complementary DNA) bank, Expressing the protein encoded by the insert
Insert the cDNA into the resulting vector, -Transform the bacterial strain with the resulting recombinant vector
And then conditions under which the desired protein can be expressed in bacteria.
Set, -Villin protein using antibodies capable of recognizing villin protein
Select recombinant clones containing specific clones
You. The construction step of the cDNA bank depends on the inserted cDNA.
Using a vector that can express the encoded protein
It is done. Claw which is particularly advantageous because of its high expression efficiency
Vector is composed of a pEX-type plasmid.
It is. Such a plasmid was provided by Stanley and Luzio.
EMBOJournal, 1984,Three, 1429-1434.
You. This document includes the promoter of the λ bacteriophage.
 P_RCro-lacZ, a fusion gene expressed under the control of
Containing plasmids have been described. This manifestation depends on temperature.
Is induced. Polynucleotide containing multiple unique restriction sites
Is inserted in each of the reading phases, and
A translation termination signal is also inserted. Insert at unique restriction site
Entered cDNA is the corresponding hybrid protein cro-
It is expressed with very high efficiency in the form of βgal-peptide.
The advantage of this hybrid protein is that it is poorly soluble and
Less proteolytic degradation in bacteria
It is easily detected immunologically by an antibody. The corresponding polypeptide is expressed in bacteria.
Insert the cDNA in the correct orientation for the gene cro-lacZ
To enter, P.N.A.S.USA, 1983,80, 31-35
Various linkers at both ends of the cDNA according to the technology of Helfmann et al.
Use a protocol that adds keys sequentially. Used resources
CarBamHI andSalCorresponds to the I cleavage site. [0041] These recombinant plasmids can be prepared using conventional techniques.
Bacterial strains that express proteins with different desired sequences
Used for quality switching. As a bacterial strain, an E. coli strain is preferable.
E. coli pop2135 containing lesser cro allele cIts857
 Is particularly preferred. Thus, high levels of villi in bacterial strains
Approximately 30,000 clones, corresponding to the mRNA of the cell expressing the protein
A cDNA bank containing the loan recombinant is obtained. The expression of the hybrid protein depends on the temperature.
Can be triggered. Therefore, the repressor of the gene cro
Treat at deactivating temperature. For this purpose, the
Incubate the strain for about 2 hours at room temperature, especially on the order of 40-42 ° C
Just cuvette. Bacterial Lysis and Proteins According to the Prior Art
Clones recovered after regenerating DNA for immunoscreening
Therefore, select. Preferably at least one type of anti-villi
The cDNA polyclonal antibody is used to screen cDNA banks.
Perform a leaning. With one or more polyclonal antibodies
Specific reacting clones against COOH-terminal epitope
Again using at least one monoclonal antibody
Screen. Anti-Polyc Against Complete Porcine Villin Protein
Anti-villin polyclonal antibodies such as nal antibodies and molecules
One or more types that can recognize the epitope located at the COOH terminus of
If you use the above monoclonal antibodies sequentially,
A leaning is performed. Screening with polyclonal antibody
Later, another polyclonal antibody, especially chicken villi
Using polyclonal antibody against COOH-terminal fragment of protein
Advantageously, a secondary screening is performed. The object of the present invention is in particular a complete villous protein.
Antibodies to Chickens and Chicken Villin Protein
Polyclonal Antibodies to COOH-terminal Peptides and CO
Two types of monoclonal antibodies against the OH-terminal epitope
Characterized in that the expression product specifically reacts with
Providing a clone containing cDNA corresponding to the villin protein
Is to provide. It is also an object of the present invention to provide a polyA
Villin protein containing an insert with a denylation site
To provide a clone containing the cDNA corresponding to
You. According to another feature of the present invention, the villous protein
The means used for quality detection are composed of monoclonal antibodies.
Is done. This feature of the invention is derived from different species
Common villous proteins accessible to monoclonal antibodies
High epitopes that secrete monoclonal antibodies
Spleen fused with myeloma cells suitable for bridoma production
Specific villous tan used to immunize animals with kidney cells
Not only specifically recognizes protein but also includes human
Specifically recognizes villous proteins derived from
Based on the fact that. Accordingly, the preferred monoclonal antibodies of the present invention
The characteristics of the body are that it has the following characteristics: Recognizing purified porcine villous protein (C)
Use Eastern blotting), -Recognize chicken villin protein (Western
Using blotting), -Villi of cell extract of a line derived from human colon adenocarcinoma HT29
Recognize proteins (using Western blotting)
for), -Recognition of rat villin protein by immunocytochemistry
(Freezing of formaldehyde-fixed rat intestinal mucosa
Liostat) using sections). A monoclonal antibody corresponding to the above definition
A preferred monoclonal antibody is chicken villi
Recognition of epitopes contained in protein “peptide HP”
Understand. Pre-isolated peptide HP and peptide HP
And 51 Kd peptide
You. The antibody is a peptide of chicken villi protein 44Kd or
Does not recognize any of the peptide 44Kd. The preferred antibody of the present invention further comprises a protein
Quality HP, especially after treatment with sodium dodecyl sulfate (SDS).
Recognizable in the raw state and contains villous proteins
Can be recognized by skin cells. This result depends on the peptide HP
Supported epitopes on other proteins in cell culture
Therefore, it is shown that it is not shielded. The present invention naturally covers these monoclonal antibodies.
Pertaining to a hybridoma that secretes. These hybrids
Advantageous techniques for producing porcine, especially purified porcine villous tan
Spleen cells of mouse immunized against protein and appropriate myelo
Of hybridoma production technology by fusion with yeast cells
Will be described later. Preferred hybrids obtained by this method
(BD-D2C3 strain) is the Collecti of Pasteur Institute in Paris.
on Nationale des Cultures de Micro-organismes (CNC
M) on April 29, 1985 with accession number no.I-440.
Was. According to an advantageous feature of the invention, the formation of antibodies
The immunizing substance used for can be expressed in large amounts in bacteria. This
The immunological substance is human villous protein DNA or a fragment thereof.
Encoded by the nucleotide sequence corresponding to
Corresponds to the peptide. According to the prior art, peptides are injected into animals.
And collect antiserum and then, if necessary, e.g.
Antibodies are recovered from antisera by column chromatography
I do. This variant of antibody production is based on the cDNA fragment defined above.
The region encoded by the fragment HP is the most immunogenic and
It is more advantageous when the region is specific to hair proteins.
You. The complementary DNA of the villin protein or the DNA
The protein encoded by the nucleotide sequence of the fragment
Quality can also be determined by competitive radioimmunoassay (direct ELISA or
Constitutes an antigen source for performing competitive ELISA. However, of course, it contains the same immunogenic sequence
All other immunizing substances can be used. For example, pepti
HP itself may be used, and in some cases,
Carrier such as calf serum albumin to enhance
Even using peptide HP pre-grafted to the molecule
Good. The preferred monoclonal antibody of the present invention
If possible, define the characteristic epitope region of the peptide HP
Obtaining will also be apparent to those skilled in the art. More accurate identification
In particular, using a method that includes the following steps:
Good. The peptide HP (or containing the desired epitope)
Polynucleic acid encoding the part of the peptide HP
Synthesizing a reotide sequence, -Nucleotides encoding the COOH terminus of the villous protein
The plasmid as defined above containing the sequence
Linearize at the external restriction site, -Linearized by an exonuclease such as the enzyme Bal31
Trimming the plasmid, Recirculating the plasmid with DNA ligase, -Can transform itself with the corresponding plasmid and
A suitable microparticle capable of expressing the insert contained in the lathmid
Transforming organisms, -Bringing the expression product of this microorganism into contact with the antibody again
You. Characterization by the final recircularized plasmid
The immunogenic peptide in the transformed expression product of the microorganism.
Repeat the above processing cycle until the detection of
You. End of each cycle of the process, especially intermediate recyclization
Final trimming to eliminate plasmid recognition ability
Before and after the sequencing of the terminal part of the plasmid
Nucleosides removed during this final trimming process
Epitope at the peptide encoded by the peptide sequence
It is possible to place loops. That is, for those skilled in the art
Obtaining the antibody by means of a chemical comprising the epitope
Is equivalent to obtaining a well-defined peptide sequence.
You. The present invention also relates to the preferred models defined above.
Polyacrylamide gel that can react with noclonal antibody
Virtually one band in electrophoresis, especially in SDS-PAGE test
Biologically Substantially Pure Human Villin Protein
Pertaining to quality. That is, human villin protein is
Treatment with a lysate, especially an aqueous solution containing a suitable detergent,
Human intestinal cell lysate purified using noclonal antibodies?
Can be extracted from In this type of purification, affinity
Preferably solid to accommodate the chromatography process
Using monoclonal antibodies immobilized on a body support
Is advantageous. For example, PHRMACIA A.G. in Sweden
Therefore, a three-dimensional mesh marketed under the trademark SEPHAROSE
Monoclonal antibody on agarose mesh
Fix by cyanogen bromide method. Accordingly, the present invention is particularly directed to human villin proteins.
To separate the villin protein-containing solution,
Contact with affinity column containing monoclonal antibody
To selectively immobilize human villin protein, and then
PH 2-4 acidic buffer or methylamine buffer
Of antigen-antibody complex by a basic pH 11 buffer
The human villin protein is recovered by the
The present invention relates to a method for dialysis against a monium buffer. Of course, the present invention also relates to human villin protein cleavage.
Pertaining to smaller molecular weight polypeptides consisting of pieces. Interruption
Human villi by fragment-specific polypeptide cleavage enzymes
It is clear to those skilled in the art that what can be obtained by protein cleavage is
Or maybe. As such a protein, for example,
LipsinStaphylococcus aureusV8 Protea
Ze, alpha chymotrypsin, marketed by BOEHRINGER
Mouse submandibular gland protease, the peptide Gly-Pro
And Gly-Ala etc.Vibrio alginolyti
-cus chemovar iophagusCollagenase. Production from human villin protein or fragment thereof
Optionally species-specific hybridomas and monochrome
It is to be understood that internal antibodies also form part of the present invention.
U. [0071] DETAILED DESCRIPTION OF THE INVENTION Another feature and advantage of the present invention is that human villi
Detection of protein, hybridoma and monoclonal antibody
Examples of specific conditions used in the production and testing of
It will be apparent from the description. Also contains cDNA portion of human villin protein
It also describes how to create clones. [0073]Example 1 Detection of Human Villin Protein Tumors from the digestive or renal parts and other parts of the organism
Villous proteins at different stages of proliferation of tumor cells and non-tumor cells
The expression of proteins was examined. BROWN and FARQUHAR, Cell,Three
6295 (1984).
Sections (frozen sections) were prepared by fixing. A section of 5 mm thickness was obtained at -25 ° C.
Placed on coated glass plate. 0.2% gelatin
Containing physiological saline phosphate buffer (PBS-gelatin) solution
After immersion, PBS-gelatin is used to detect villous proteins.
Rabbit anti-villin protein antiserum 50 diluted 1/800 in protein
Treat the sections with a solution containing
Incubated. Next, the sections were cut in PBS-gelatin for 10 minutes each.
Washed twice. Next, Rhodamine (Nordic Immun, The Netherlands)
rabbit anti-rabbit labeled
IgG was added and sections were incubated at room temperature in the presence of these anti-IgGs.
Incubated for minutes. Then cut the sections in PBS-gelatin
Lens thoroughly cleaned and immersed in Plan Neo-fluar oil
Fluorescence microscope equipped with a set of appropriate filters (ZEIS
S light microscope). Control tissue and tumor cells of other origin
Was similarly processed. The following results were observed. When the tissue is derived from adenocarcinoma of the colon, 12
Eleven samples of the villin protein expression
Positive. Human tumors of other origin, such as lymphoma
Mesenteric nodal lymphoma and metastasis of pancreas, esophagus, etc.
Various types of carcinomas of other origins other than
No protein was expressed. Observations generally indicate that one of the treated tumors
A Close Correlation between the Intestinal Origin and Villin Protein Expression
It turned out that there was a clerk. Clearly against tumors of another origin
In a number of tests conducted in
The expression of the protein was negative. It should be noted that in tissues of intestinal origin,
Villin protein is always irrespective of the degree of cell growth
Is to be observed. In the above test, a polyclonal antibody was used.
Was. Uses preferred monoclonal antibody deposited with CNCM
The result was more obvious. High secreting these monoclonal antibodies
Examples of conditions for isolating the bridoma are described below. Also, EL
ISA-type immunoassay to determine the presence of villous protein
The following describes how to provide favorable detection conditions for diagnosis.
It is described below. [0084]Example 2: Villous proteinqualityMonochrome against
Preparation of internal antibody (A) Immunization program −Balb / C o mouse: 6-8 weeks old −antigen: Purified porcine villous protein (10mg / ml) (SDS
In the presence of polyacrylamide gel (10%) after electrophoresis
Homogeneous band of 95Kd protein), 10mM imida
Sol, 75 mM HCl, 1 mM EGTA, 0.1 M DTT, 50%
Serol. -program First day: 50/50 emulsion (PBS-Freund complete adjuvant)
IP (peritoneal cavity) of 50 μg of pure villin protein in
Inside) injection, 14th: 50 μg of villous protein (PBS-Freund incomplete)
Booster IP injection in adjuvant emulsion), 21st: 50 μg of villous protein (PBS-Freund incomplete)
Booster IP injection in adjuvant emulsion), Day 28: IM of 20 μg villous protein dissolved in PBS (muscle
Intrameat) injection, 29th: IV (intravenous) injection of 10 μg villi dissolved in PBS
Shooting, 32nd: Fusion. (B) Fusion (I) Parent cell (a)Myeloma cells Sp2 / O-Ag14 (8 Azaguanine resistant), DMEM 10% FCS medium
Sterile culture in. (B)Spleen cells Origin: Hyperimmune mouse Balb / C against porcine villous protein
Splenocytes. (II)) Details by Kohler G. and Milstein C.
Cell fusion method (fused cells secreting antibodies with specificity
Continuous culture of Nature, 1975, 256, 495) -50% polyethylene glycol in serum-free DMEM medium
Solution (PEG1000-Merck9729) under sterile conditions.
Fuse in serum DMEM medium. After counting two types of parent cells
The suspension of myeloma cells in culture is
Mix 1: 5 with the solution. Contact for 2 minutes and 30 seconds. -Stop the action of PEG by diluting with complete DMEM medium
To stop. -Finalize cell suspension in selective DMEM-HAT medium
Dilution (2 × 10^FiveCells / ml). Dispense into COSTAR24 wells containing -1 ml wells (C
OSTAR Tissue Culture Cluster 24, Cat.No.3524, CO
STAR 205 Groadway, Cambridge, Ma, USA). (III)Clone selection The ability to secrete antibodies to purified porcine villin protein
To identify the clone. (C) Selection method (I) ELISA test: In this test, the
An anti-villin protein monoclonal antibody can be demonstrated. -Fixation of antigen: For support (ELISA plaque)
Fix the antigen. Concentration: 50 mM, pH 8 potassium phosphate bath
5 μg / ml in buffer. Maintain at 4 ° C. overnight. -Saturation: Saturated with PBS-Tween20-BSA. -incubationI: Undiluted hybrid
3 hours at 4 ° C. with dormer supernatant. -incubationII: β-galactosida
2 h at 4 ° C. with mouse-labeled mouse anti-IgG. For washing between each step, 0.1% of PBS-
Use Tween20. -Substrate: 0.1 M, pH 7.0 phosphate buffer,
Ten^-3M MgSO_Four, 2 × 10^-3M MnSO_Four, 2 × 10^-3M anhydrous bird
Triplex Magnesium (Merck8409)
O-Nitrophenyl-β-galactopyranoside (Sigma
 N11-27). [0102]ReadingOD (optical density) reading at 414 nm. The OD of 10 times the OD of the background noise
A clone with the given hybridoma supernatant was selected. (II)) Western Blotting(Burnette.Anal.B
iochem. 1981, 112, 195-203) This test will help to ensure that
Simultaneous human and chicken villous proteins
Secretes porcine anti-villi monoclonal antibodies recognizable by
Clones can be selected. [0105]Various test stages (a) Chicken intestinal mucosal cell extract and villous protein
HT29 (human colon adenocarcinoma) cell extract
Perform electrophoresis on a acrylamide gel. (B) Separated by polyacrylamide gel
The protein is electrotransferred to nitrocellulose paper. (C) Nitroses to which cell proteins have been transferred
Place the lulose paper on the hybridoma previously selected by ELISA.
Incubate with clearing at 4 ° C. overnight. (D) Peroxidase-labeled mouse
Incubate with anti-IgG for 1 hour at room temperature. (E) Substrate: -10 mg of diaminobenzidine tetrahydrochloride, -20 ml 0.1 M TrisHCl pH 7.6, -0.2 ml of 1% H_TwoO_Two. [0110] Washing between each step was performed using neonatal calf blood.
Use Qing-PBS-Triton X100. (F) Positive reaction: antigen-monoclonal antibody
When the reaction occurs, a maroon band (molecular weight 95 Kd) is rapidly formed.
Appear. (D) Cloning of selected hybrids -Limiting dilution method: macrophages bound to the bottom of the well
(4 week old Balb / C mouse origin) (96-well plaque
Positive) so that one cell is distributed per well
Diluted in selective media. [0113] The selection medium whose conditions have been adjusted is added. Selecting positive clones by the method described above
You. Recloning if necessary. (E) Preparation of ascites -PRISTANE (Aldrich, 2,6,10,14-tetra-
(Methylpentadecane)
Inject a loan. 1-2 × 10 per mouse⁶Injection of cells
I do. Including clonal cells grown after 10-15 days
The ascites is collected from the mice. The anti-villi monoclonal antibody titer of ascites was 1 mg
/ Ml higher. -Freezing of selected clones is 10% FCS-5% DM
Performed in DMEM medium with SO. Store in liquid nitrogen at -176 ° C. [0121]Direct ELISA for villous protein quantification −Adsorption of purified IgG： Concentrated ascites BD-D_2C3From ELISA
Adsorb to plaque. Ion exchange chromatography (DEAE-Tris
(Acrylic) and hydroxyapatite columns
The body was purified. Concentrate to a predetermined concentration. Incubation at 37 ° C for 2 hours and then at 4 ° C overnight
To Wash with 0.1% PBS / Tween20. -Plaque saturation 30 min in the presence of PBS / 0.1% Tween20 / 0.4% BSA
Incubate. -Addition of antigen The concentration range of purified villin protein in the presence of 0.4% BSA
Reduce from 5 μg / ml to 1 or 0.1 ng / ml. Incubate at 4 ° C. for 3 hours. Wash with 0.1% PBS / Tween20. -Add purified IgG: With β-galactosidase
Rabbit polyclonal bound to villin protein
Add IgG purified from antibody sera. Incubate at 4 ° C. for 2 hours. Wash in 0.1% PBS / Tween20. -Galactosidase detection 20 ml of 0.1 M phosphate buffer buffer, pH 7, 10^-3M MgSO
_Four, 2 × 10^-FourM MnSO_Four, 2 × 10^-3M EDTA (Merck840
9) 20 mg of p-nitrophenyl- using a 4 m / ml solution in
 Add β-D-galactopyranoside. Incubate at 37 ° C. for X hours. Read the optical density at 414 nm. [0136]Example 3: Human clinical observation Very small amounts in cell extracts (0.
5 ng) with the sensitivity to detect villous protein
Quantification of villous protein in serum using ELISA test
A measurement was performed to detect the effective amount. That is, villi
The protein is released by healthy and tumor cells in the gastrointestinal tract.
Because it is an intracellular protein that is expressed,
Under physiological conditions, necrosis of cells containing
It was found that the protein was released into the vasculature. According to tests on the blood donation population (n = 190),
Villin protein is not detected in the majority of serum (n = 168)
Turned out to be. However, for a small number of individuals (n = 15)
Indicates detectable villous protein (5-10 ng / ml)
did. This value is the level of villous protein that is not a pathological sign.
You can think. Finally, for some individuals (n = 7)
Higher levels of villous protein (50-100ng / ml)
Were present. So-called “healthy” individuals (3.6%)
Additional laboratory tests of the gastrointestinal tract (fiberscope,
Ronoscope). The results obtained for various gastrointestinal pathologies are described below.
These are summarized in the table below. Detectable amounts of villous protein in the patient's serum
Diseases whose quality is detected:Gastrointestinal malignant pathology : -Colon cancer, -Gastric cancer. [0140]Gastrointestinal benign pathology: -Choriomas, -Crohn's disease, -Hemorrhagic proctitis, -Duodenal bulb ulcer, gastric ulcer, esophageal ulcer. It should be noted that polyadenomas of the digestive tract (Poly
Conversely, by not releasing this protein into the blood
is there. [0142] Some further conclusions can be drawn from this examination.
You. 1 / Amount of villous protein in blood and tumor size
It may be considered that there is no correlation between the magnitude or the stage of its progress. 2 / This protein is used for post-operative follow-up.
When measured, the rate in serum decreases after complete removal. this
Conversely, if tumor cells remain (incomplete removal, recurrence,
Metastasis) Villin protein levels are maintained or increased
You. According to this study, villin protein was
Normally not detected in the examiner's serum,
Cancer or kidney cancer) or inflammatory or digestive mucosa
Is a benign ulcer of the gastrointestinal tract that produces necrotic processes (Crohn's disease, RC
Villin protein in patients with H disease, ulcers)
It turns out that it can be done. The quantification of villous protein in blood was also good.
For monitoring inflammatory diseases (ulcers of the digestive tract and inflammatory diseases)
Turned out to be important. On the other hand, villous tan in blood
In order to track protein, cancer progression is performed with the antibody of the present invention.
CEA (Carcinoembryonic Anti-
It is possible to use another tumor label, such as
You. These current tumor labels are 1 / specific to the affected organ
Deficiency, 2 / Observed during various benign pathologies
Has a point. [0147]Example 4: cD corresponding to human villous protein
Preparation of NA Preparation of mRNA MRNA is prepared from the human colon adenocarcinoma cell line HT29. Ul
Guanidium chloride method by lrich et al., Science, 1977, 1
96, 1313-1319, to extract RNA. Aviv and Le
oligo dT-cellulose column chromatography with der
Acad.Sci. USA, 1972, Proc.69, 1408-141
2, by mRNA poly A⁺Is purified. [0148]Preparation of cDNA Fiddes & Goodman Method, Nature, 1979,281, 351-3
56, the first strand and the second strand of cDNA
Is prepared. Helfmann et al., Proc. Natl. Acad. Sci. U
SA, 1983,80, 31-35, the linker Sa at the 3 'end
BamHI is added sequentially to the I and 5 'ends. A cDNA van containing approximately 30,000 recombinant clones
Get [0150]vector Plasmid pEX1-3 (Stanley and Luzio, EMBO Journal,
1984,Three, 1429-1430, 1984). these
The vector is a λ bacteriophage promoter whose expression
Data P_RCro-LacZ, a fusion gene under the control of
Derived from the containing plasmid. Multiple unique restriction sites
Inserted into the 3 'end of LacZ gene
did. In addition, three readings of transcription and translation termination signals
Inserted into the phase. The plasmid digested with the restriction enzymes BamHI and SalI
CDNA was inserted into each of the Smid pEX. So these are all
Is oriented in the same direction with respect to the gene CroLacZ. [0152]Transformation O.Raibaud for Nucleic Acid Research
Use the particularly constructed E. coli pop2135 strain as a bacterial strain.
You. Manipulating according to the following formula,CI85
7And the promoter P_R2.3 kb of lambda phage containingBgl
The II fragment is cloned into the BamHI site of the polylinker.
You. [0154] Embedded image The thus obtainedEcoThe RI fragment was ligated with pOM4
OneEcoCloned into the RI site and cloned into the chromosome of strain C600
(Gene,292231-241). [0156] ProximalaroBCotransfer of label and P1 '
This structure was transformed into MM294 chromosome (F 'endA
thi hsdR). E. coli pop2135 is orientedma
IT,CI857, P_R,maIPOIs obtained. Transform E. coli pop2135 with the recombinant plasmid
To convert, use a technique that uses rubidium such as Hanahan.
Jutsu, J. Mol. Biol., 1983,166, 557-580.
This strain contains the cro repressor allele cIts857.
Including. The number of recombinants obtained was 10^Three~Ten^Fourng cDNA
Of the order. [0159]Selection of clones by immunodetection The obtained recombinant clone is filtered with a nitrocellulose filter.
Smear on. High after 2 hours incubation at 42 ° C
Induces synthesis of brid protein. Fine by SDS
Lysis and reconstitution of proteins in the absence of methanol
Analyze clones by immunoscreening after birth
You. For protein regeneration, Burnett's technology, Anal.Bioch
em., 1981,112, 195-203. First, the port for the complete porcine villous protein was
Screening banks with clonal antibodies
You. Next, the COOH-terminal fragment of chicken villin protein
Polyclonal antibody against the COOH-terminal region of the molecule.
Monoclonal antibodies that recognize epitopes
Body (BDD_TwoC_ThreeAnd IID_ThreeH₉) And using secondary script
Perform training. The clone pEXZ-V19 has a 510 base pair
Including The coding portion has 330 base pairs. This part
Following the minute is a 200 base pair noncoding region. The cloned cDNA was a human villous protein.
It encodes the 95 COOH-terminal amino acids of
It is equivalent to about 1/10 of protein (molecular weight: 95Kd).

──────────────────────────────────────────────────の Continuation of the front page (51) Int.Cl. ⁶ Identification number Office reference number FI Technical indication (C12P 21/08 C12R 1:91) (73) Patent holder 594198569 Ansteuteu Nacional do la Sante et Dou La Luciersi-yu-Medical France 75001 Paris, Lille-du-Torbiac, 101 (72) Inventor Daniel Lebar France, 92330 Saw, Are Dou Trevis, 23 (72) Inventor Brigitte d'Eudewe, France, 75005 Paris, Lille-Brunville, 9 (72) Inventor Silvi Robineau, France, 92170 Bambu, Lille Deissy, 54 (72) Inventor Monique Alpin France, 75014 Paris, Lille Lori, 9 (72) Inventor Erik Plan Over France, 75003 Paris, reusable-o-mail, 26 (72) inventor Arufuonsu Garcia France, 75015 Paris, Riyu de Woo Birofure, 3

Claims (1)

(57) [Claims] Have the following characteristics, namely - it recognizes purified porcine villi protein, - recognizing the villi protein of chicken, - recognizing the villi protein of cell extract derived systems adenocarcinoma HT29 human colon, - immune A monoclonal antibody against villin protein, which recognizes rat villus protein cytochemically. 2. The antigenic site contained in the C-terminal sequence (779-854), ie, the formula: Can be recognized, regardless of whether the head portion is isolated or present inside a 51Kd peptide contained in the chicken villin protein. The monoclonal antibody according to claim 1, wherein the monoclonal antibody can recognize a part. 3. For hybridoma with accession number CNCM I-440
2. The model according to claim 1, wherein
Noclonal antibodies.